Produktion af Adeno-Associerede Virus Vektorer i Cell Stakke til prækliniske undersøgelser i store dyremodeller
Summary
Her giver vi en detaljeret procedure for storstilet produktion af AAV-vektorer af forskningskvalitet ved hjælp af klæbende HEK 293 celler dyrket i cellestakke og affinitetskromatografirensning. Denne protokol giver konsekvent >1 x 1013 vektorgenomer/mL, hvilket giver vektormængder, der er passende til store dyreforsøg.
Abstract
Adeno-associerede virus (AAV) vektorer er blandt de mest klinisk avancerede genterapi vektorer, med tre AAV genterapier godkendt til mennesker. Klinisk udvikling af nye anvendelser af AAV indebærer overgangen fra små dyremodeller, såsom mus, til større dyremodeller, herunder hunde, får og ikke-menneskelige primater. En af begrænsningerne ved at administrere AAV til større dyr er kravet om store mængder af høj titervirus. Mens suspension celle kultur er en skalerbar metode til AAV vektor produktion, få forskningslaboratorier har udstyret (f.eks bioreaktorer) eller ved, hvordan man producerer AAV på denne måde. Desuden er AAV titere ofte betydeligt lavere, når de produceres i suspension HEK 293 celler sammenlignet med klæbende HEK293-celler. Beskrevet her er en metode til fremstilling af store mængder af høj-titer AAV ved hjælp af celle stakke. En detaljeret protokol for titering AAV samt metoder til validering vektor renhed er også beskrevet. Endelig præsenteres repræsentative resultater af AAV-medieret transgeneudtryk i en fåremodel. Denne optimerede protokol til storstilet produktion af AAV-vektorer i klæbende celler vil gøre det muligt for molekylærbiologilaboratorier at fremme afprøvningen af deres nye AAV-behandlinger i større dyremodeller.
Introduction
Genterapi udnytte adeno-associeret virus (AAV) vektorer har gjort store fremskridt i løbet af de sidste tre årtier1,2. Påviste forbedringer i en bred vifte af genetiske sygdomme, herunder medfødt blindhed, hæmofili og sygdomme i bevægeapparatet og centralnervesystemet, har bragt AAV-genterapi i spidsen for klinisk forskning3,4. I 2012 godkendte Det Europæiske Lægemiddelagentur (EMA) Glybera, en AAV1-vektor, der udtrykker lipoprotein lipase (LPL) til behandling af LPL-mangel, hvilket gør det til den første markedsføringstilladelse til en genterapibehandling i enten Europa eller USA5. Siden da har yderligere to AAV-genterapier, Luxturna6 og Zolgensma7, modtaget FDA-godkendelse, og markedet forventes at ekspandere hurtigt i løbet af de næste 5 år med så mange som 10-20 genterapier forventes i 20258. Tilgængelige kliniske data viser, at AAV genterapi er en sikker, veltolereret, og effektiv modalitet gør det til en af de mest lovende virale vektorer, med over 244 kliniske forsøg med AAV registreret med ClinicalTrials.gov. Den stigende interesse for kliniske anvendelser, der involverer AAV-vektorer, kræver robuste og skalerbare produktionsmetoder for at lette evalueringen af AAV-behandlinger i store dyremodeller, da dette er et kritisk skridt i den translationelle pipeline9.
For AAV vektor produktion, de to vigtigste krav er AAV genomet og capsid. Genomet af vildtype (wt)-AAV er enkeltstrenget DNA, der er ca. 4,7 kb i længden10. Wt-AAV-genomet omfatter omvendte terminalgentninger (ITR’er), der findes i begge ender af genomet, som er vigtige for emballagen, og rep- og cap-generne 11. Rep og cap gener, der er nødvendige for genom replikation, samling af viral capsid, og indkapsling af genomet i viral capsid, fjernes fra det virale genom og leveres i trans til AAV vektor produktion12. Fjernelsen af disse gener fra det virale genom giver plads til terapeutiske transgener og alle de nødvendige regulatoriske elementer, herunder promotoren og polyA-signalet. ITR’erne forbliver i vektorgenomet for at sikre korrekt genomreplikation og viral indkapsling13,14. For at forbedre kinetikken i transgene udtryk, AAV vektor genomer kan manipuleres til at være selv komplementære, hvilket mindsker behovet for konvertering fra enkelt-strandede til dobbeltstrenget DNA-konvertering under AAV genom replikation, men reducerer kodning kapacitet til ~ 2,4 kb15.
Ud over AAV genom design, capsid serotype udvælgelse bestemmer væv og celle tropofi af AAV vektor in vivo2. Ud over vævstrofi har forskellige AAV-serotyper vist sig at vise forskellige genekspressions kinetik16. For eksempel klassificerede Zincarelli et al.17 forskellige AAV-serotyper til lavtryks serotyper (AAV2, 3, 4, 5), moderate udtryksrotyper (AAV1, 6, 8) og højtryks serotyper (AAV7 og 9). De kategoriserede også AAV-serotyper i slow-debut udtryk (AAV2, 3, 4, 5) eller hurtig debut udtryk (AAV1, 6, 7, 8 og 9). Disse divergerende troper og genekspression kinetik skyldes aminosyre variationer i capsid proteiner, capsid protein formationer, og interaktioner med værtscelle receptorer / co-receptorer18. Nogle AAV capsids har yderligere gavnlige egenskaber såsom evnen til at krydse blod – hjerne barrieren efter intravaskulær administration (AAV9) eller opholde sig i langlevende muskelceller for holdbare transgene udtryk (AAV6, 6.2FF, 8, og 9)19,20.
Dette papir har til formål at detaljere en omkostningseffektiv metode til fremstilling af høj renhed, høj titer, forskning-grade AAV vektorer til brug i prækliniske store dyremodeller. Produktionen af AAV ved hjælp af denne protokol opnås ved hjælp af dobbelt-plasmid transfection i klæbende menneskelige embryonale nyre (HEK) 293 celler dyrket i celle stakke. Desuden beskriver undersøgelsen en protokol for heparin sulfat affinitet kromatografi rensning, som kan bruges til AAV serotyper, der indeholder heparin-bindende domæner, herunder AAV2, 3, 6, 6.2FF, 13, og DJ21,22.
En række emballagesystemer er tilgængelige til produktion af AAV vektorer. Blandt disse har brugen af to-plasmid co-transfection systemer, hvor Rep og Cap gener og Ad helper gener (E1A, E1B55K, E2A, E4orf6, og VA RNA) er indeholdt i en plasmid (pHelper), har nogle praktiske fordele i forhold til den fælles tre-plasmid (tredobbelt) transfection metode, herunder reducerede omkostninger for plasmid produktion23,24 . AAV-genomplasmiden, der indeholder transgeneudtrykskassetten (pTransgene), skal flankeres af ITR’er og må ikke overstige ~4,7 kb i længden. Vektor titer og renhed kan blive påvirket af transgene på grund af potentielle cytotoksiske virkninger under transfection. Vurdering af vektor renhed er beskrevet heri. Vektorer produceret ved hjælp af denne metode, som giver en 1 x 1013 vg/ mL for hver, blev evalueret i mus, hamstere og får dyremodeller.
Tabel 1: Sammensætning af nødvendige løsninger. Nødvendige oplysninger, herunder procentdele og mængder, af komponenter, der er nødvendige for forskellige løsninger i hele protokollen. Klik her for at downloade denne tabel.
Protocol
Representative Results
Discussion
Produktionen af rekombinante AAV (rAAV) vektorer beskrevet i dette papir bruger fælles materialer, reagenser, og udstyr findes i de fleste af de molekylærbiologiske forskningslaboratorier og faciliteter. Dette papir giver mulighed for høj kvalitet in vitro og in vivo kvalitet rAAV, der skal produceres af læseren. Frem for alt er denne protokol for rAAV-produktion sammenlignet med mere kedelige protokoller, der involverer rensning af cæsiumchlorid, effektiv og undgår brugen af ultracentrifugering. …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Amira D. Rghei, Brenna A. Y. Stevens, Sylvia P. Thomas, og Jacob G. E. Yates var modtagere af Ontario Veterinary College Student Stipends samt Ontario Graduate Stipendier. Amira D. Rghei var modtager af en Mitacs Accelerate Studentship. Dette arbejde blev finansieret af canadian Institutes for Health Research (CIHR) Project Grant (#66009) og et Collaborative Health Research Projects (NSERC partnered) grant (#433339) til SKW.
Materials
| 0.22 μm filter | Millipore Sigma | S2GPU05RE | |
| 0.25% Trypsin | Fisher Scientific | SM2001C | |
| 1-Butanol | Thermo Fisher Scientific | A399-4 | CAUTION. Use under a laminar flow hood. Wear gloves |
| 10 chamber cellstack | Corning | 3271 | |
| 1L PETG bottle | Thermo Fisher Scientific | 2019-1000 | |
| 30% Acrylamide/Bis Solution | Bio-Rad | 1610158 | |
| 96-well skirted plate | FroggaBio | FS-96 | |
| Adhesive plate seals | Thermo Fisher Scientific | 08-408-240 | |
| Ammonium persulfate (APS) | Bio-Rad | 161-0700 | CAUTION. Use under a laminar flow hood. Wear gloves |
| Blood and Tissue Clean up Kit | Qiagen | 69506 | Use for DNA clean up in section 4.6 of protocol |
| Bromophenol blue | Fisher Scientific | B392-5 | CAUTION. Use under a laminar flow hood. Wear gloves |
| Cell Culture Dishes | Greiner bio-one | 7000232 | 15 cm plates |
| Culture Conical Tube | Thermo Fisher Scientific | 339650 | 15 mL conical tube |
| Culture Conical Tube | Fisher Scientific | 14955240 | 50 mL conical tube |
| Dulbecco's Modified Eagle Medium (DMEM) with 1000 mg/L D-glucose, L-glutamine | Cytiva Life Sciences | SH30022.01 | |
| ECL Western Blotting Substrate | Thermo Fisher Scientific | 32209 | |
| Ethanol | Greenfield | P016EA95 | Dilute ethyl alcohol(95% vol) to 20% for section 3.7.4 and 70% for section 6.1.1.1 |
| Fetal Bovine Serum (FBS) | Thermo Fisher Scientific | SH30396.03 | |
| Glacial acetic acid | Fisher Scientific | A38-500 | CAUTION. Use under a laminar flow hood. Wear gloves |
| Glycerol | Fisher Scientific | BP229-1 | |
| Glycine | Fisher Scientific | BP381-500 | |
| HBSS with Mg2+ and Ca2+ | Thermo Fisher Scientific | SH302268.02 | |
| HBSS without Mg2+ and Ca2+ | Thermo Fisher Scientific | SH30588.02 | |
| HEK293 cells | American Tissue Culture Collection | CRL-1573 | Upon receipt, thaw the cells and culture as described in manufacturer’s protocol. Once cells have been minimally passaged and are growing well, freeze a subfraction for future in aliquots and store in liquid nitrogen. Always use cells below passage number 30. Once cultured cells have been passaged more than 30 times, it is recommended to restart a culture from the stored aliquots |
| HEK293 host cell protein ELISA kit | Cygnus Technologies | F650S | Follow manufacturer’s instructions |
| Heparin sulfate column | Cytiva Life Sciences | 17040703 | |
| Kimwipe | Thermo Fisher Scientific | KC34120 | |
| L-glutamine (200 mM) | Thermo Fisher Scientific | SH30034.02 | |
| Large Volume Centrifuge Tube Support Cushion | Corning | CLS431124 | Support cushion must be used with large volume centrifuge tubes uless the centrifuge rotor has the approriate V-bottom cushions |
| Large Volume Centrifuge Tubes | Corning | CLS431123-6EA | 500 mL centrifuge tubes |
| MgCl2 | Thermo Fisher Scientific | 7791-18-6 | |
| Microcentrifuge tube | Fisher Scientific | 05-408-129 | 1.5 mL microcentrifuge tube, sterilize prior to use |
| Molecular Grade Water | Cytiva Life Sciences | SH30538.03 | |
| N-Lauroylsarcosine sodium salt | Sigma Aldrich | L5125 | CAUTION. Wear gloves |
| NaCl | Thermo Fisher Scientific | BP35810 | |
| Optimem, reduced serum medium | Thermo Fisher Scientific | 31985070 | |
| Pasteur pipets | Fisher Scientific | 13-678-20D | Sterilize prior to use |
| PBS (10x) | Thermo Fisher Scientific | 70011044 | Dilute to 1x for use on cells |
| Penicillin-Streptomycin Solution | Cytiva Life Sciences | SV30010 | |
| pHelper plasmid | De novo design or obtained from plasmid repository | NA | |
| Pipet basin | Thermo Fisher Scientific | 13-681-502 | Purchase sterile pipet basins |
| Polyethylene glycol tert-octylphenyl ether (Triton X-100) | Thermo Fisher Scientific | 9002-93-1 | CAUTION. Wear gloves |
| Polyethylenimine (PEI) | Polyscience | 24765-1 | Follow manufacturer’s instructions to produce a 1L solution. 0.22μm filter and store at 4°C |
| Polypropylene semi-skirted PCR Plate | FroggaBio | WS-96 | |
| Polysorbate 20 (Tween 20) | Thermo Fisher Scientific | BP337-100 | CAUTION. Wear gloves |
| polyvinylidene difluoride (PVDF) membrane | Cytiva Life Sciences | 10600023 | Use forceps to manipulate. Wear gloves. |
| Primary antibody | Progen | 65158 | |
| Protein Ladder | FroggaBio | PM008-0500 | |
| Proteinase K | Thermo Fisher Scientific | AM2546 | |
| pTrangene plasmid | De novo design or obtained from plasmid repository | NA | Must contain SV40 polyA in genome to be compatible with AAV titration in section 5.0 |
| Pump tubing | Cole-Parmer | RK-96440-14 | Optimize length of tubing and containment of virus in fractions E1-E5 |
| RQ1 Dnase 10 Reaction Buffer | Promega | M6101 | Use at 10x concentration in protocol from section 4.0 |
| RQ1 Rnase-free Dnase | Promega | M6101 | |
| Sample dilutent | Cygnus Technologies | I700 | Must be purchased separately for use with HEK293 host cell protein ELISA kit |
| Secondary antibody, HRP | Thermo Fisher Scientific | G-21040 | |
| Skim milk powder | Oxoid | LP0033B | |
| Sodium dodecyl sulfate (SDS) | Thermo Fisher Scientific | 28312 | CAUTION. Use under a laminar flow hood. Wear gloves |
| Sodium hydroxide (NaOH) | Thermo Fisher Scientific | SS266-4 | |
| SV40 polyA primer probe | IDT | Use sequence in Table X for quote from IDT for synthesis | |
| Tetramethylethylenediamine (TEMED) | Thermo Fisher Scientific | 15524010 | CAUTION. Use under a laminar flow hood. Wear gloves |
| Tris Base | Fisher Scientific | BP152-5 | |
| Trypan blue | Bio-Rad | 1450021 | |
| Ultra-Filter | Millipore Sigma | UFC810024 | Ultra-4 Centrifugal 10K device must be used, as it has a 10000 molecular weight cutoff |
| Universal Nuclease for cell lysis | Thermo Fisher Scientific | 88702 | |
| Universal qPCR master mix | NEB | M3003L | |
| Whatman Paper | Millipore Sigma | WHA1001325 | |
| β-mercaptoethanol | Fisher Scientific | 21985023 | CAUTION. Use under a laminar flow hood. Wear gloves |
| CAUTION: Refer to the Materials Table for guidelines on the use of dangerous chemicals. |
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